The applicant proposes to study the role of the alpha3 Na,K-ATPase isoform in the heart focusing on testing the overall hypothesis that the alpha3 Na,K-ATPase isoform is adapted to function under conditions of stress since it has a lower Na affinity than alpha1 and alpha2 isoforms. The specific objectives are: 1) To examine how forced overexpression of alpha3 affects ionic homeostasis and contractility in genetically manipulated mouse heart cells and perfused hearts; 2) To determine the effect of preventing the Na,K-ATPase isoform switch by forcing overexpression of alpha3 in hearts of 30-day-old transgenic mice; 3) To develop methods to suppress alpha3 pumps in neonatal rat heart cells and determine the functional consequences of such suppression. Methods: Structure-function relationships will be probed by studying both heart cells from transgenic mice, and cultured rat heart cells in which the alpha3/alpha1 isoform expression ratio has been manipulated via antisense technology. Intracellular cation levels and fluxes in living cells will be studied with fluorescence spectroscopy. Then the effects of ouabain on contractility in normal and transgenic hearts will be compared using videomicroscopy and isolated heart perfusion.